Topoisomerase II; Daunorubicins/Doxorubicins

Daunorubicin (0-256 μg/mL, 30 min) inhibits the synthesis of DNA and RNA in sensitive and resistant Ehrlich ascites tumor cells[2].
Daunorubicin (7 nM-1.9 μM, 72 h) exhibits chemosensitivity in Molt-4 cells and L3.6 cells[3][4].
Daunorubicin (0.4 μM, 48 h) causes necrosis and apoptosis in L3.6 cells[4].
Daunorubicin (0.4 μM, 120 min) causes ROS generation in L3.6 cells[4].
Daunorubicin (2 μM, 24 h) induces autophagy in K562 cells (myeloid cell line)[6].

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Treatment with daunorubicin (DNR) at a dose of 2 µM for 24 hours induced autophagy in myeloid leukemia cell lines (K562 and KG1a), as evidenced by increased conversion of LC3-I to LC3-II, upregulation of the lysosomal marker LAMP-2, and downregulation of p62/SQSTM1 protein levels in Western blot analysis. This effect was dose-dependent. [5]
The induction of autophagy by daunorubicin (2 µM) was further confirmed by increased immunofluorescence staining of LC3 and LAMP-2, and by enhanced autophagic flux when co-treated with the lysosomal inhibitor chloroquine. [5]
Treatment with daunorubicin (2 µM) significantly increased the expression of autophagy-related genes ATG14, GABARAPL1, and SMPD1 at the protein and/or mRNA level in K562 cells. The expression of ATG9A was not significantly changed by DNR treatment. [5]
Daunorubicin treatment (2 µM) completely prevented the proliferation of K562 cells over a 4-day culture period. This antiproliferative effect was partially prevented when autophagy was inhibited by co-treatment with 3-methyladenine (3-MA), suggesting that DNR decreases cell growth partly by inducing autophagy. [5]
In K562 cells, treatment with lower doses of daunorubicin (0.1 µM and 0.5 µM) for 72 hours led to a downregulation of cell survival, as measured by cell counting and a luminescent cell viability assay. [5]

Daunorubicin (intravenous injection, 3 mg/kg, three times at 48 h intervals.) causes nephrotoxicity and cardiotoxicity in rats[5].
Daunorubicin (intraperitoneal injection, 10 mg/kg) causes sister chromatid exchanges in mice[7].

Daunorubicin inhibits of both DNA and RNA syntheses in HeLa cells over a concentration range of 0.2 through 2 μM.

Daunorubicin significantly inhibits the biosynthesis of DNA and RNA macromolecules when administered to leukemic cells isolated from patients with acute lymphocytic leukemia.

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Western Blot Analysis for Autophagy Markers: Cells (e.g., K562, KG1a) were treated with various doses of daunorubicin (e.g., 2 µM) or vehicle (DMSO) for specified times (e.g., 24 hours). To assess autophagic flux, cells were co-treated with chloroquine (CQ, 10 µM) for the last 4-16 hours of incubation. After treatment, cells were harvested, washed with cold phosphate-buffered saline (PBS), and lysed in cold lysis buffer containing protease inhibitors. Following incubation on ice and centrifugation, protein concentration in the supernatant was determined. Equal amounts of protein (30-40 µg) were separated by electrophoresis on polyacrylamide gels and transferred to membranes. The membranes were blocked and then incubated overnight at 4°C with primary antibodies against LC3B, LAMP-2, p62/SQSTM1, ATG9A, ATG14, GABARAPL1, and β-actin (loading control). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescence detection system. [5]
Immunofluorescence Staining for Autophagy Markers: After treatment with daunorubicin and/or other agents, cells were transferred to slides using a cytospin centrifuge. Cells were fixed with paraformaldehyde, permeabilized with saponin, and blocked. They were then incubated overnight at 4°C with primary antibodies against LC3 and LAMP-2. After washing, cells were incubated with fluorescent dye-conjugated secondary antibodies (e.g., Alexa Fluor 488, Alexa Fluor 568) and the nuclear stain DAPI. Stained cells were washed, post-fixed, mounted, and visualized using fluorescence microscopy. [5]
Cell Viability/Proliferation Assay (Cell Counting): K562 cells were treated with daunorubicin (e.g., 0.1 µM, 0.5 µM, 2 µM) and/or other compounds (e.g., 3-MA, miR-15a-5p mimic/inhibitor). Viable cells were counted at various time points (e.g., days 1, 2, 3, 4) in the presence of Trypan Blue dye using a hemocytometer. Only unstained (viable) cells were counted. [5]
Luminescent Cell Viability Assay: K562 cells were seeded in 96-well plates and treated with daunorubicin (e.g., 0.1 µM, 0.5 µM) for 72 hours. After treatment, a single reagent (CellTiter-Glo® Reagent) was added directly to the culture well, mixed, and incubated. The resulting luminescent signal, which is proportional to the amount of ATP present (an indicator of metabolically active cells), was measured using a luminometer. [5]
Reverse Transcription Quantitative PCR (RT-qPCR) for Gene Expression: Total RNA was extracted from treated and control cells. For mRNA analysis, RNA was reverse transcribed into cDNA. Quantitative PCR was performed using gene-specific primers for ATG9A, ATG14, GABARAPL1, SMPD1, and the housekeeping gene RPLP0. The relative expression of target genes was calculated using the comparative Ct method. [5]

Male Sprague-Dawley rats eight weeks of age are employed. Two more weeks are spent acclimating and keeping the animals in quarantine before the experiments begin. Day 0: A single intravenous injection of Daunorubicin (3 mg/kg) is given to each animal. To achieve an accumulative dose of 9 mg/kg, daunorubicin is given in three equal injections spaced 48 hours apart over the course of one week. It is well known that this dosage will cause nephrotoxicity and cardiotoxicity. As a control, age-matched rats (group Control; n=5) are injected with corresponding volumes of 0.9% NaCl. Twenty-two DNR-treated rats were split into two groups at random and given either a vehicle (group Daunorubicin; n = 12) or Telmisartan (10 mg/kg/day; group Daunorubicin+Telmisartan; n = 10). Telmisartan dosage is determined by referencing an earlier study. Commencing the day of Daunorubicin administration, Telmisartan is administered for an additional 5 weeks after Daunorubicin administration is stopped, for a total of 6 weeks of administration. Previous reports are the basis for choosing this study duration. Body weight (BW) and protein concentrations are measured on day 41 after rats are individually housed in metabolic cages for a 24-hour urine collection period. Following the completion of the six-week study period, kidney tissue is extracted from the rats and used for semi-quantitative immunoblotting and immunohistochemical analyses.
Rats from Sprague-Dawley

Absorption, Distribution and Excretion
After 90 minutes of infusion of the liposomal formulation at a dose of 44 mg/m², the time to peak concentration (tmax) of daunorubicin was found to be 2 hours, and the peak plasma concentration (cmax) was 24.8 μg/mL. Daunnorubicin is excreted via the liver. 40% of daunorubicin is excreted in bile, and 25% is excreted in the urine in its active form (daunorubicin or daunorubicin alcohol). In the liposomal formulation, only 9% of the active molecule is excreted in the urine. The reported steady-state volume of distribution (VOD) of daunorubicin in the liposomal formulation is 1.91 L/m². The mean VOD of the liposomal formulation is 6.6 L. The clearance of daunorubicin is 68.4 mL/h/m², as measured with the liposomal formulation. Note: Liposome encapsulation significantly affects the functional properties of the drug, making it different from the unencapsulated drug. Furthermore, different liposomal drug products may differ in the chemical composition and physical morphology of the liposomes. These differences can significantly affect the functional properties of liposomal drug products. Encapsulating daunorubicin citrate in liposomes significantly alters the drug's pharmacokinetics. Compared to conventional intravenous formulations (i.e., non-encapsulated drugs), its pharmacokinetic characteristics are significantly altered, leading to reduced distribution in peripheral tissues, increased distribution in Kaposi's sarcoma lesions, and decreased plasma clearance. Daunorubicin hydrochloride is highly irritating to tissues and therefore must be administered intravenously. In patients with HIV-associated Kaposi's sarcoma, following a single intravenous injection of 40 mg/m² liposomally encapsulated daunorubicin citrate, the average peak plasma concentration of daunorubicin (primarily bound to liposomes) was approximately 18 μg/mL 30–60 minutes after infusion. This results in a higher peak plasma concentration of daunorubicin after intravenous administration of liposomally encapsulated daunorubicin citrate compared to conventional (non-encapsulated) daunorubicin hydrochloride. In one study, patients with disseminated malignancies who received a single intravenous dose of 80 mg/m² of unencapsulated daunorubicin had a peak plasma concentration of 0.4 μg/mL; while in patients with solid tumors (including Kaposi's sarcoma) who received a single intravenous dose of 80 mg/m² of liposomal daunorubicin, the peak plasma concentration was approximately 44 μg/mL (approximately 100 times higher than in patients receiving the same dose of unencapsulated daunorubicin). The area under the plasma concentration-time curve (AUC) of liposomal daunorubicin was approximately 36 times higher than that of conventional daunorubicin hydrochloride. Following intravenous administration of liposomal daunorubicin, peak plasma concentration and AUC generally increased linearly with increasing dose (dose range 10–80 μg/mL). For more complete data on absorption, distribution, and excretion of daunorubicin (18 studies in total), please visit the HSDB records page.

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Metabolism/Metabolites
Daunorubicin hydrochloride is primarily metabolized in the liver and other tissues, mainly by cytoplasmic aldehyde-ketone reductases, to produce daunorubicin alcohol, the main metabolite with antitumor activity. Following injection of unencapsulated daunorubicin, approximately 40% of the drug remains in plasma as daunorubicin alcohol, and approximately 60% remains in this form within 4 hours.
Following intravenous injection of daunorubicin citrate liposomes, only low concentrations of daunorubicin alcohol were detected in plasma. In patients with HIV-associated Kaposi's sarcoma who received 40 mg/m² liposomal daunorubicin intravenously, the AUC of daunorubicin alcohol accounted for only 2% of the total AUC of daunorubicin. Further metabolism from glycosidic bond reduction cleavage produces an aglycone, which has little or no cytotoxic activity and is demethylated by microsomal enzymes, conjugated to sulfate and glucuronic acid. Metabolites identified in human urine include daunorubicin alcohol, daunorubicin aglycone, demethyldeoxydaunorubicin aglycone, demethyldeoxydaunorubicin aglycone-4-O-sulfate, demethyloxydaunorubicin aglycone-4-O-glucuronide, and deoxydaunorubicin aglycone glucuronide. It is extensively metabolized, initially into active alcohol metabolites; further metabolized by hepatic microsomes into inactive aglycones and demethylated glucuronides and sulfate conjugates. Hepatic elimination pathway: Approximately 25% of the administered dose of daunorubicin hydrochloride is excreted in its active form in the urine, and approximately 40% is excreted in the bile. Half-life: 18.5 hours. The terminal half-life of daunorubicin has been determined to be 18.5 hours (±4.9). The terminal half-life of the major active metabolite, daunorubicin alcohol, has been determined to be 26.7 hours (±12.8). Pharmacokinetic studies showed that the mean elimination half-life of liposomal daunorubicin was 22.1 hours, while the official label data from the U.S. Food and Drug Administration (FDA) was 31.5 hours. Following rapid intravenous injection of conventional daunorubicin hydrochloride, the total plasma concentrations of daunorubicin and its metabolites decreased in three phases, while the plasma concentrations of unchanged daunorubicin decreased in two phases. The plasma half-life of unencapsulated daunorubicin averaged 45 minutes in the initial phase and 18.5 hours in the terminal phase. One hour after injection of unencapsulated daunorubicin, the dominant form of the drug in plasma was the active metabolite daunorubicin alcohol, with a mean terminal plasma half-life of 26.7 hours. The apparent elimination half-life of DaunoXome (daunorubicin citrate liposomal injection) was 4.4 hours, significantly shorter than that of daunorubicin, and may represent the distribution half-life.

Hepatotoxicity
Daunorubicin combined with other chemotherapy drugs can cause elevated serum enzymes in some patients, depending on the dosage and other medications used. ALT elevations during daunorubicin treatment are usually asymptomatic and transient, resolving spontaneously without dose adjustment. In many cases, it is difficult to attribute abnormal liver function to daunorubicin due to concurrent exposure to other potentially hepatotoxic drugs. Currently, there is no conclusive evidence that daunorubicin treatment causes acute, clinically significant, specific liver injury with jaundice. However, high-dose daunorubicin combined with other antitumor drugs has been associated with cases of hepatic sinusoidal obstruction syndrome, which typically presents with right upper quadrant pain 10 to 30 days after infusion, followed by weight gain, ascites, and abnormal liver function. There have been cases of death due to liver failure, but most patients recover within 1 to 3 months of onset. Probability Score: E (Unproven but suspected cause of clinically significant liver injury). Daunorubicin induces necrotic cell death, manifested by 96% cell uptake of PI after 24 hours and 93% after 48 hours. The ROS generation induced by daunorubicin may be related to its cytotoxic and necrotic effects.

[1]. Activity of topoisomerase inhibitors daunorubicin, idarubicin, and aclarubicin in the Drosophila Somatic Mutation and Recombination Test. Environ Mol Mutagen. 2004;43(4):250-7.

[2]. Inhibition of DNA and RNA synthesis by daunorubicin in sensitive and resistant Ehrlich ascites tumor cells in vitro. Cancer Res. 1972 Jun;32(6):1307-14.

[3]. Melanin inhibits cytotoxic effects of Doxorubicin and Daunorubicin in MOLT 4 cells. Pigment Cell Res. 2003 Aug;16(4):351-4.

[4]. An effective in vitro antitumor response against human pancreatic carcinoma with paclitaxel and Daunorubicin by induction of both necrosis and apoptosis. Anticancer Res. 2004 Sep-Oct;24(5A):2617-26.

[5]. Telmisartan prevents the progression of renal injury in daunorubicin rats with the alteration of angiotensin II and endothelin-1 receptor expression associated with its PPAR-γ agonist actions. Toxicology. 2011 Jan 11;279(1-3):91-9.

[6]. MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin. Int J Mol Sci. 2021 May 13;22(10):5153.

[7]. Doxorubicin suppresses chondrocyte differentiation by stimulating ROS production. Eur J Pharm Sci. 2021 Dec 1;167:106013.

According to an independent committee of scientific and health experts, daunorubicin may be carcinogenic.
Anthracycline antibiotic. An anticancer drug.
Daunorubicin is a natural product found in Actinomadura roseola. It is both an antitumor drug and a bacterial metabolite. It is an anthracycline antibiotic belonging to the tetrabenzoquinone, paraquinone, and aminoglycoside antibiotics. It is the conjugate base of daunorubicin(1+). It is derived from the hydride of tetrabenzoquinone.
A highly toxic anthracycline aminoglycoside antitumor drug, isolated from strains such as Streptomyces peucetius, used to treat leukemia and other tumors.
Daunorubicin is an anthracycline topoisomerase inhibitor. The mechanism of action of daunorubicin is as a topoisomerase inhibitor.
Daunorubicin is an anthracycline antibiotic with antitumor activity, used to treat acute leukemia and AIDS-related Kaposi's sarcoma. Transient elevations in serum enzymes and bilirubin during daunorubicin treatment are rare, but have not been found to be associated with clinically significant acute liver injury with jaundice. Daunnorubicin has been reported in Streptomyces, Brassica napus, and other microorganisms with relevant data. Daunnorubicin is an anthracycline antitumor antibiotic with similar therapeutic effects to doxorubicin. It exerts its cytotoxic activity through topoisomerase-mediated DNA interactions, thereby inhibiting DNA replication and repair, as well as RNA and protein synthesis. Daunnorubicin is only present in individuals who have used or taken the drug. It is a highly toxic anthracycline aminoglycoside antitumor drug, isolated from strains such as Streptomyces peucetius, and used to treat leukemia and other cancers. [PubChem] Daunorubicin possesses antimitotic and cytotoxic activities, with multiple mechanisms of action: daunorubicin forms a complex by inserting itself between DNA base pairs and inhibits topoisomerase II activity by stabilizing the DNA-topoisomerase II complex, thereby preventing the rejoining portion of the topoisomerase II-catalyzed ligation-rejoining reaction. Daunorubicin is a highly toxic anthracycline aminoglycoside antitumor drug, isolated from strains such as Streptomyces peucetius, and used to treat leukemia and other tumors. See also: Daunorubicin hydrochloride (note moved here).

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Drug Indications
For remission induction therapy in adults with acute non-lymphocytic leukemia (myeloid, monocytic, erythroid), and for remission induction therapy in children with acute lymphoblastic leukemia. Adults. Daunorubicin, in combination with cytarabine, is used to treat newly diagnosed treatment-related acute myeloid leukemia (t-AML) or acute myeloid leukemia with myelodysplastic associated changes (AML-MRC) in adults and children aged 1 year and older.
Mechanism of Action

Daunorubicin exerts its antimitotic and cytotoxic activities through several proposed mechanisms of action: Daunorubicin forms a complex with DNA by inserting between base pairs and inhibits the activity of topoisomerase II by stabilizing the DNA-topoisomerase II complex, thereby preventing the rejoining portion of the topoisomerase II-catalyzed ligation-rejoining reaction.
Daunorubicin is an antitumor antibiotic. Daunorubicin has antimitotic and cytotoxic activities. Daunorubicin forms a complex with DNA by inserting between base pairs. Daunorubicin inhibits the activity of topoisomerase II by stabilizing the DNA-topoisomerase II complex, leading to single-strand and double-strand breaks in DNA. Daunorubicin may also inhibit polymerase activity, affect gene expression regulation, and participate in DNA free radical damage. Although daunorubicin exhibits maximum cytotoxicity in the S phase, it is not a cell cycle-specific drug. Daunnorubicin also possesses antibacterial and immunosuppressive effects. Anthracyclines are important drugs in many chemotherapy regimens for treating various tumors. One of the main mechanisms of action of anthracyclines is through DNA damage caused by the inhibition of topoisomerase II. Enzymatic detoxification of anthracyclines is a key factor determining anthracycline resistance. The natural product daunorubicin is a toxic anthracycline antibiotic analog that can be reduced to the less toxic daunorubicin alcohol by the AKR1B10 enzyme. The AKR1B10 enzyme is overexpressed in most cases of smoking-related squamous cell carcinoma (SCC) and adenocarcinoma. Furthermore, the AKR1B10 enzyme has also been found to be overexpressed in human liver cancer, cervical cancer, and endometrial cancer in samples from uterine cancer patients. AKR1B10 enzyme expression is also associated with postoperative tumor recurrence in cervical cancer and keratinization in squamous cell carcinoma, and is considered to have potential as a target for intervention in colorectal cancer cells (HCT-8) and a diagnostic marker for non-small cell lung cancer. This article elucidates the mechanism of action of daunorubicin and proposes a method to enhance its efficacy by modulating AKR1B10 activity. In this study, researchers used ATP-depleting agents cyanide, azide, or dinitrophenol to inhibit energy-dependent transport processes and observed even higher accumulations of daunorubicin compared to cyclosporine A (CsA). Similar patterns were observed in various P-gp-negative human cancer cell lines. Furthermore, the observed cyanide effect was independent of the expression of multidrug resistance-associated protein (MRP) mRNA, the only known member of the ABC membrane transporter family capable of effluxing daunorubicin. These results suggest that in many cases of acute myeloid leukemia (AML), daunorubicin accumulation is regulated by one or more novel energy-dependent processes distinct from P-gp or MRP. The authors hypothesize that this novel drug transport mechanism may influence the response of AML patients to daunorubicin and other therapeutic agents. Daunorubicin inhibits DNA synthesis and blocks DNA-directed RNA polymerase. At doses that do not interfere with nucleic acid synthesis, it can prevent cell division.

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Daunorubicin (DNR) is an anthracycline antibiotic and a cornerstone drug for induction chemotherapy in acute myeloid leukemia (AML). It is most commonly used in combination with cytarabine in the standard “7+3” induction regimen. [5]
This study explored the clinical significance of dose-enhanced daunorubicin therapy, noting that higher doses have been reported to improve remission and survival rates in certain AML patient subgroups, particularly those under 65 years of age or with poor cytogenetic prognosis, without significantly increasing toxicity. [5]
This study focuses on the resistance mechanism of daunorubicin (DNR). Studies have shown that DNR can induce autophagy in acute myeloid leukemia (AML) cell lines (K562, KG1a), which contributes to the cytotoxic effect of the drug. microRNA miR-15a-5p confers chemotherapeutic resistance by inhibiting DNR-induced autophagy. [5]

C27H29NO10

527.53

527.179

C, 61.48; H, 5.54; N, 2.66; O, 30.33

20830-81-3

30323

Dark Red Solid powder

1.6±0.1 g/cm3

770.0±60.0 °C at 760 mmHg

155ºC

419.5±32.9 °C

0.0±2.8 mmHg at 25°C

1.692

2.92

5

11

4

38

960

6

O=C(C(C(OC)=CC=C1)=C1C2=O)C3=C2C(O)=C(C[C@@](O)(C(C)=O)C[C@@H]4O[C@@]5([H])C[C@H](N)[C@H](O)[C@H](C)O5)C4=C3O

STQGQHZAVUOBTE-VGBVRHCVSA-N

InChI=1S/C27H29NO10/c1-10-22(30)14(28)7-17(37-10)38-16-9-27(35,11(2)29)8-13-19(16)26(34)21-20(24(13)32)23(31)12-5-4-6-15(36-3)18(12)25(21)33/h4-6,10,14,16-17,22,30,32,34-35H,7-9,28H2,1-3H3/t10-,14-,16-,17-,22+,27-/m0/s1

(7S,9S)-9-acetyl-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione

Daunomycin HCl; RP 13057; Rubidomycin; RP-13057; RP13057; Daunomycin hydrochloride; daunomycin HCl; daunorubidomycine; US brand names: Cerubidine; Rubidomycin; Foreign brand names: Cerubidin; Daunoblastin; Daunoblastina; Ondena; Rubilem; Abbreviations: DNM; DNR; DRB; Code names: FI6339; RP13057

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)